During Phase I work IA successfully applied evanescent sensing technology to the study of aryl hydrocarbon receptor (AhR)-mediated processes and provided discrimination between beneficial and harmful AhR ligands. When biosensors were used to assess the interaction of AhR with ligands, accessory proteins, and dioxin response element (DRE) distinctive sensorgrams emerged for each type of compound. Under Phase II work applications of this will be developed for the research and the environmental and food-testing markets. This will be achieved through the Specific Aims: (1) Optimize and standardize reagents and protocols; (2) Specify the detailed nature of the instrument; (3) Establish data interpretation methods that quantify and reliably distinguish between receptor agonists, antagonists and partial agonists; (4) Improve biosensor fluorometer instrument; (5) Improve design and manufacturing of biosensor cartridges; (6) Validate the system and methods on 30-50 compounds; and (7) Obtain regulatory approval for use of the AhR biosensor for dioxin screening. Existing methods for studying these mechanisms are cumbersome and time consuming, and for cell-based methods, compound degradation by cellular metabolism and instability of cell cultures significantly affects results. During Phase II, the biosensor instrument and reagents will be optimized and prototyped and will be used to assess the impact of ligands on the mechanisms affecting cross-talk between endocrine disrupting compounds at the level of receptor-DNA interactions and to predict the effects of compounds based on mechanism-based sensor evaluation. It is anticipated that parameterization of sensorgrams will provide criteria for discrimination between AhR agonists, antagonists, mixed agonist/antagonists and compounds which do not interact with AhR. The resulting biosensor, reagents and protocols will be validated by using them to categorize 30-50 blind coded compounds. This will initially be done at 1A, Inc. The proposal incorporates a time and resources extension which will support similar testing in 4 independent laboratories. Results will be submitted to the ICCVAM validation process. The biosensor system will also be tested for identification of dioxin-like activity in real-world samples obtained from the Great Lakes watershed. Once the system is validated it will have particular value in the food testing arena. Recent regulatory legislation by the EU requires that foods be certified free of dioxin contamination. This will cause the $6.5 billion industry which exports U.S. food to the EU to be in need of a cost effective, rapid, on-site screening test such as will be provided by this project. [unreadable] [unreadable]